The Effects And Potential Mechanisms Of High Glucose On Function Of Vascular Smooth Muscle Cells In Atherosclerosis

BackgroundsThe past few decades have witnessed a dramatic worldwide increase in the incidence of diabetes mellitus that has been driven by changes in lifestyle and escalating obesity rates.Diabetes mellitus is a major independent risk factor for cardiovascular diseases(CVDs)and individuals with diabetes are 2 to 3 times more likely to die from a heart attack or stroke than people without diabetes,even after controlling for other cardiovascular risk factors.Both type 1 and type 2 diabetes have been shown to accelerate the development of atherosclerosis(AS),the underlying cause of most myocardial infarctions.It is the primary mission for basic carvascular research to seek for a more effective way to treat and prevent AS formation and development in diabetes.There is considerable evidence that many biochemical pathways adversely affected by hyperglycemia including glucose oxidation,the formation of advanced glycation end-products(AGEs),and activation of polyol pathways,are associated with the generation of reactive oxygen species(ROS),ultimately leading to increased oxidative stress in a variety of tissues in diabetes.After intimal injury by ROS,different cell types,including endothelial cells(ECs),platelets,and inflammatory cells release mediators,such as growth factors and cytokines that induce multiple effects.The inflammatory response simulates migration and proliferation of VSMCs that become intermixed with the area of inflammation to form an intermediate lesion.These responses continue uninhibited and is accompanied by accumulation of new extra cellular matrix(ECM).Thus VSMCs proliferation and migration is the key part of AS formation in diabetes.The migratory and proliferative activities of VSMCs are regulated by growth promoters such as platelet derived growth factors(PGF),endothelin-1(ET-1),thrombin,fibroblast growth factor(FGF),interleukin-1(IL-1)and inhibitors such as,heparin sulfates,nitric oxide(NO),transforming growth factor(TGF)-beta.The matrix metallo proteinases(MMPs)could also participate in the process of VSMCs migration.MMPs could catalyze and remove the basement membrane around VSMCs and facilitate contacts with the interstitial matrix.This could promote a change from quiescent,contractile VSMCs to cells capable of migrating and proliferating to mediate repair.Cell proliferation is dependent on the cell cycle proceeding,which is highly ordered and tightly regulated by cyclins,cyclin-dependent kinases(CDKs),and CDK inhibitors(CKIs).After a vascular injury,VSMCs are stimulated to divide in response to mitogens,and exiting the G1 phase and entering the S phase.Cyclin D1-CDK4 and cyclin E-CDK2 predominantly act in sequence during the G1/S transition and are required for cell cycle progression through this period.Many pharmacological agents,including anti-proliferative agents of VSMC,as well as anti-thrombotic and anti-platelet agents,could alter one or more regulatory events in the cell cycle resulting in blockade of cell cycle progression,thereby suppressing cell proliferation.Poly(ADP-ribose)polymerase 1(PARP-1)is a nuclear protein,mainly known for DNA repair by catalyzing poly ADP-ribosylation of itself and other chromatin-associated proteins.Besides,PARP-1 is implicated in a broad variety of cellular functions,including gene transcriptional regulation,cell cycle progression,inflammation,energy metabolism and cell migration.In diabetes,ROS and reactive nitrogen species(RNS)are potent activators of PARP-1 as a result of their ability to damage DNA.PARP-1 activation plays a key role in the pathogenesis of various diabetic complications such as vasculopathy,cardiomyopathy,retinopathy and neuropathy.However,its specifc role in neointimal hyperplasia in diabetes and precise molecular mechanisms have not been clarifed.Objectives1.To explore the effects of high glucose on PARP-1 expression in HASMCs;2.To identify the role of PARP-1 on HASMCs proliferation and migrtion,and explore the underlying mechanisms.3.To identify the role of PARP-1 on neointima formation in diabetic mice.Methods1.Cell culture and treatmentHuman aortic smooth muscle cells(HASMCs,ATCC)were grown in Smooth Muscle Cell Medium containing 2%foetal bovine serum(FBS).HASMCs(passages 5-8)were exposed to 5 mM D-glucose(normal control,NC),5 mM D-glucose+27.5 mM mannose(osmotic control,OC),33 mM D-glucose(high glucose,HG),33 mM D-glucose+5?M PARP-1 inhibitor PJ34(HG+PJ34)for 72 h.2.Cell proliferation assayProliferation was analysed by using Cell-LightTM EdU assay according to the manufacturer's directions.3.Cell cycle assayCell cycle analysis was performed by flow cytometry.4.Cell migration assayHASMC migration assays were performed in Transwell chambers.5.Gelatin zymographyCell culture supernatants were electrophoresed in a 10%polyacrylamide gel containing 1 mg/ml gelatin.Lytic bands of gelatin digestion represented MMP2 and MMP9 activity.6.Western blot analysisProteins were extracted from treated HASMCs,and the expressions of PARP-1,PAR,MMP2,MMP9,PCNA and ?-actin were determined,7.Diabetic Mice Model and Carotid Artery LigationC57BL/6J wild-type mice and C57BL/6J/PARP-1-/-mice were were randomized into the following three groups:(?)wild-type non-diabetic group(the control group),(?)wild-type diabetic group,(?)PARP1-/-diabetic group.Type 1 diabetes mellitus was initiated by the administration of 5 daily intraperitoneal injections of 50 mg/kg streptozotocin(STZ);Mice with blood glucose levels of>300 mg/dL after 2 weeks were included in diabetic cohorts.Then,ligation of the left carotid artery was performed.Animals were anesthetized as above 3 weeks after surgery and killed by perfusion with 4%paraformaldehyde.The left carotid arteries and aorta of mice were embedded in paraffin and serially sectioned(5 ?m).8.Human tissue harvestHuman coronary arteries used in this study were obtained from Qilu hospital of Shandong University.The patient data were reviewed to exclude tissue from patients with malignant tumors,acute inflammatory disease,rheumatism and other systemic disease.The distal part of left circumflex arteries were embedded in paraffin and serially sectioned(5 ?m).We observed the sections under the microscope,and divided them into two group:the normal group and arteries with neointima hyperplasia.9.Histology and immunohistochemistryThe sections were stained with hematoxylin and eosin(H&E)to observe the extent of neointimal hyperplasia.For immunohistochemistry,the sections were labelled with PARP-1,MMP2,PPM9 and PCNA.10.Statistical analysisAll experiments were repeated at least three times,and data were presented as the mean ±S.E.M.Statistical analysis was carried out using ANOVA followed by Tukey's post hoc test(GraphPad Software,USA).P<0.05 was considered significant.Results1.High glucose up-regulates PARP-1 expression in HASMCsHigh glucose significantly increased PARP-1 expression compared with the control,and the maximium effects were observed at 33mM after 72-hour stimulation.2.High glucose promotes HASMCs proliferation via PARP-1 activationIn Edu incorporation assay,high glucose markedly stimulated DNA synthesis,while PARP-1 inhibition largely reversed these effects.3.PARP-1 activation promotes G1-S transition in HASMCsWe analysed cell cycle progression by using flow cytometry analysis.High glucose stimulation significantly promoted G1-S transition in HASMCs,while there was a significantly higher number of cells arrested in the G1 phase with PARP-1 inhibition than without.Furthermore,the expression of proliferating cell nuclear antigen(PCNA),which regulates programmes governing the G1-S transition,was reduced to nearly non-stimulated level by PJ34 treatment.4.High glucose promotes HASMCs migration via PARP-1 activationTranswell migration assays were used to evaluate the effects of PARP-1 on the migration of HASMCs,PARP-1 inhibition significantly suppressed high glucose-stimulated cell migration.Moreover,we observed that PARP-1 inhibition markedly attenuated high glucose-induced increase in MMP-2 and MMP-9 protein expression,which are in accordance with the altered MMPs activity in gelatin zymography.5.The role of PARP-1 in augmented neointimal hyperplasia in diabetic miceWild-type diabetic mice exhibited dramatically augmented neointimal thickening of carotid artery compared with the control group,and it was largely attenuated in PARP-1-/-diabetic mice.Immunohistochemical analysis revealed that the expression level of PARP-1,PCNA,MMP-2 and MMP-9 was significantly increased in the neointima of wild-type diabetic mice compared with the control,while PARP-1 knockdown markedly decreased their intensity.The results were in consistent with those obtained from immunostaining in tunica media of aorta.6.The roles of PARP-1 in neointima hyperplasia of human coronaryImmunohistochemical analysis showed a higher PARP-1 abundance in neointima of stenotic coronary arteries compared with the normal ones.Reasonablely,the immunoreactivity of PCNA,MMP-2 and MMP-9 was also more evident in hyperplastic neointima.Conclusion1.The expression of PARP-1 was up-regulated in high-glucose-stimulated HASMCs and arteries of diabetic mice.2.PARP-1 promoted HASMCs proliferation by inducing G1-S transition.3.PARP-1 promoted HASMCs migration by up-regulating MMP2/9 expression and activity.4.PARP-1 promoted neointimal hyperplasia in diabetic mice.IntroductionAlthough the prevalence of diabetes and its vascular complications are increasing,the pathogenesis of diabetes and its complications have not been clearly understood.There is growing evidence supporting the role of 'metabolic memory' in diabetic complications and metabolic memory plays a critical role in the development of vascular complications in diabetic patients.Metabolic memory is the phenomenon of diabetic vascular stresses,which can persist after glucose normalization in diabetic patients due to the early hyperglycaemic environment.Hyperglycaemia appears to be remembered in organs such as the vessels,heart,kidney and eyes.Increasing studies show that epigenetics may be the underlying mechanisms,which can explain metabolic memory.Epigenetic processes play a critical role in regulating tissue-specific gene expression and hence alterations in these processes may induce long-term changes in gene function and metabolism,which can persist throughout the course of diseases.Epigenetic mechanisms,including DNA methylation,histone methylation,histone acetylation,activate multiple signal transduction pathways and regulate related gene expression,involving blood vessels,heart,kidney and eyes.Therefore,it can increase the susceptibility of macrovascular complications such as atherosclerosis,dilated cardiomyopathy and microvascular complications.5-methylcytosine is considered to be the fifth base of DNA as-through its non-random distribution along the genome-it constitutes part of the epigenetic chromatin modifications that control gene expression patterns.The genome methylation pattern is bimodal:the methylated cytosines are scattered throughout the genome,whereas the unmethylated residues are mainly located within particular regions termed CpG islands.The 37,000 CpG islands in the mouse genome represent 1-2%of the DNA and are generally located in the 59 promoter regions of the housekeeping genes,sometimes overlapping the coding region to variable extents.Although their sequence is enriched in CpG dinucleotides,the best substrates for DNA methyltransferase(DNMT)activity,the CpG islands are mainly unmethylated and the associated genes are actively transcribed;transcription is inhibited when these regions undergo methylation.Over the past decade,PARP-1 has been implicated in multiple pathways that regulate gene expression,including effects on chromatin dynamics,transcriptional regulation and epigenetics.The poly(ADP-ribosyl)ation of histiones leading to open chromatin conformation at DNA damage sites was the first indication to the function of PAR as an epigenetic modification.Lodhi et al.have demonstrated PARP-1 as a genome-wide epigenetic memory mark in mitotic chromatin.They report that PARP-1 establishes stable epigenetic marks at the transcription start sites in metaphase chromatin and these marks are a prerequisite for transcriptional restart afer mitosis.PARP-1 activity also epigenetically regulates mitochondrial DNA repair and transcription.Moreover,a series of studies have shown that PARP-1 can affect the genomic DNA methylation patern via DNMT-1,both by regulating its expression as well as actvity.A genome-wide association study suggested that inhibition of PARylation leads to hypermethylation of a substantial number of genes,as well as hypomethylation at a fraction of genes,revealing that the interplay between PARP-1 and DNA methylation is very complex.In the first section,we have already reported that PARP-1 expression was markedly increased in high glucose-stimulated HASMCs,promoting cell proliferation and migration.Now we will explore the molecular mechanism downstream PARP-1.Objectives1.To screen out the downdream molecules changed significantly when PARP-1 inhibition,and verify one molecule which is most likely involved in the proliferation and migration of VSMCs;2.To define the role of this downdream molecule in VSMCs proliferation and migration;3.To explore the regulation mechanism of PARP-1 on this molecule.Methods1.Microarray analysisHASMCs were treated with PARP-1 inhibitor PJ34 or PBS,and total RNA was extracted for microarray assay.Differentially expressed genes were detected with the GeneChip Primeview Human Gene Array(Affymetrix,CA,USA).2.Adenovirus vectors infection and cell treatmentHASMCs were infected with adenovirus vector mediated tissue factor pathway inhibitor-2 overexpression(OE-TFPI-2)and the negative control(OE-NC),or adenovirus vector containing short-hairpin RNAs(shRNAs)to suppress TFPI-2 expression via RNAi(shRNA-TFPI-2)and the negative control(shRNA-NC)at a multiplicity of infection(MOI)of 100,and mediun was changed overnight.Then the cells were cultured for 72h,or exposed to 33mM D-glucose or 27.5mM mannose+5mM D-glucose for 72h.3.Western blot analysisProteins were extracted from HASMCs,and the expressions of TFPI-2,MMP2,MMP9,PCNA and ?actin were determined.4.Cell proliferation assayProliferation was analysed by using Cell-LightTM EdU assay according to the manufacturer's directions.5.Cell cycle assayCell cycle analysis was performed by flow cytometry.6.Cell migration assay HASMC migration assays were performed in Transwell chambers.7.Gelatin zymographycell culture supernatants were electrophoresed in a 10%polyacrylamide gel containing 1 mg/ml gelatin.Lytic bands of gelatin digestion represented MMP2 and MMP9 activity.8.Detection of TFPI-2 methylationGenomic DNA was extracted from HASMCs and modified with sodium bisulfte.DNA methylation levels were determined using the Sequenom MassARRAY platform..9.Diabetic Mice Model and Carotid Artery LigationC57BL/6J wild-type mice and C57BL/6J/PARP-1-/-mice were were randomized into the following three groups:(?)wild-type non-diabetic group(the control group),(?)wild-type diabetic group,(?)PARP1-/-diabetic group.Type 1 diabetes mellitus was initiated by the administration of 5 daily intraperitoneal injections of 50 mg/kg streptozotocin(STZ);Mice with blood glucose levels of>300 mg/dL after 2 weeks were included in diabetic cohorts.Then,ligation of the left carotid artery was performed.Animals were anesthetized as above 3 weeks after surgery and killed by perfusion with 4%paraformaldehyde.10.ImmunohistochemistryLeft carotid arteries of mice were embedded in paraffin and serially sectioned(5?m).The sections were labelled with TFPI-2.11.Statistical analysisAll experiments were repeated at least three times,and data were presented as the mean ±S.E.M.Statistical analysis was carried out using ANOVA followed by Tukey'spost hoc test(GraphPad Software,USA).P<0.05 was considered significant.Results1.PARP-1 downregulates TFPI-2 gene expression The genome-wide microarray assay showed a significant upregulation of TFPI-2 with PARP-1 inhibition,which takes part in the biological behavior of cell proliferation and migration.To verify the microarray analysis data,we examined TFPI-2 expression by Western blot.High glucose stimulation significantly reduced TFPI-2 expression,which was reversed by PJ34 treatment.Immunohistochemical analysis also showed that TFPI-2 level in the neointima of carotid artery was markedly lower in wild-type diabetic mice compared with the control,which was reversed in PARP-1-/-diabetic mice,implying the role of PARP-1 on TFPI-2 expression.2.TFPI-2 negatively regulates proliferation and migration of HASMCsWe firstly explored the effects of TFPI-2 on the proliferation and migration of HASMCs in normal environment.Edu incorporation assays showed that TFPI-2 overexpression significantly suppressed DNA synthesis,while TFPI-2 knockdown significantly stimulated DNA synthesis.Transwell migration assays showed that TFPI-2 overexpression markedly suppressed the migration of HASMCs,while TFPI-2 knockdown stimulated cell migration.We then observed that TFPI-2 significantly down-regulated PCNA,MMP-2 and MMP-9 protein expression,which was in accordance with the altered MMPs activity in gelatin zymography.3.TFPI-2 inhibits high glucose induced proliferation and migration of HASMCsUnder high glucose condition,the stimulated cell proliferation,cell cycle progression and migration were suppressed by TFPI-2 overexpression.Moreover,TFPI-2 overexpression markedly attenuated high glucose-induced increase in PCNA,MMP-2 and MMP-9 protein expression,as well as MMPs activity.4.PARP1 induced hypermethylation of TFPI-2 promoterTo determine whether decreased TFPI-2 expression in high glucose-stimulated HASMCs is due to promoter methylation,the methylation levels of the TFPI-2 promoter CpG island were examined using the MassARRAY platform(Sequenom).The methylation status at CpG₃ site was much higher in high glucose-stimulated HASMCs than in the control groups,while the TFPI-2 overexpression significantly decreased the methylation levles at CpG₃,4 sites.5.TFPI-2 promotor hypermethylation accounts for its low expressionWe then used 5-Aza-dC,a DNMT inhibitor,to verify whether TFPI-2 hypermethylation accounts for its low expression.In line with our expectations,DNMT inhibition significantly reversed high glucose-induced decrease in TFPI-2 expresion,as well as increase in PCNA,MMP2 and MMP9 expression.Conclusions1.PARP-1 downregulates TFPI-2 gene expression in high glucose-stimulated HASMCs and neointima of diabetic mice.2.TFPI-2 negatively regulates proliferation and migration of HASMCs.3.PARP1 induces hypermethylation of TFPI-2 promoter which may account for its low expressionIntroductionPPAR? is a member of the nuclear receptor superfamily of ligand-inducible transcription factors and regulates multiple pathways involved in the development of diabetes and cardiovascular diseases(CVDs).It regulate transcription of target genes by heterodimerizing with the retinoid X receptor(RXR)and binding to PPAR response elements(PPRE)of regulatory promoter regions of target genes.PPAR? is expressed by macrophages,endothelial cells,and vascular smooth muscle cells,and it regulates gene expression of key proteins involved in lipid metabolism,vascular inflammation,and proliferation contributing to atherogenesis and postangioplasty restenosis.Pharmacological ligands to PPAR?,troglitazone,rosiglitazone,and pioglitazone all inhibit VSMCs proliferation in vitro at drug levels that are achieved when patients are given these agents in anti-diabetic doses.Examination of the mechanism by which thiazolidinediones inhibit VSMCs proliferation indicates that PPAR? activation attenuated the mitogen-induced degradation of p27Kip1,which inhibits cyclins/CDKs activity and phosphorylation of Rb,resulting in G1 arrest.Furthermore,PPAR? activation inhibits VSMCs migration by transrepression of Ets-1 and subsequent inhibition of MMPs production.Advanced glycation end products(AGEs),a heterogeneous group of complex structures,form nonenzymatically when reducing sugars react with free amino groups on proteins,lipids,or nucleic acids.AGEs formation and accumulation,processes of normal aging driven by hyperglycemia,occur at an accelerated rate in diabetic patients and may participate in the pathogenesis of diabetic vascular disease.The receptor for AGEs(RAGE)is found on the cell surface of endothelial cells,monocytes,macrophages,vascular smooth muscle cells,pericytes,mesangial cells,and other cells that play important roles in the onset and progression of diabetic complications.Except for direct regulation of cell cycle progression and MMPs expression,PPAR? agonist has been shown to inhibit AGEs induced VSMCs proliferation,migration and diabetic neointima hyperplasia by downregulating RAGEs expression.In section ?,we have reported that TFPI-2 can inhibit the activity of MMPs,suppressing the migration and proliferation of VSMCs.But whether this inhibition is indirect or direct is controversial.Studies have shown that TFPI-2 can inhibit the expression of MMP-2,9 by promoting phosphorylation of peroxisome proliferator-activated receptor gamma(PPAR?),thereby reducing the degradation of ECM,which slows the occurrence of atherosclerotic lesions and increases the stability of plaque.Thus PPAR-y might be one of the downstream molecule of TFPI-2.Ginsenosides,the pharmacological active phytochemicals of ginseng,have been demonstrated to exert beneficial effects on diabetes and cardiovascular diseases(CVDs).Ginsenosides mostly existing as enantiomeric pairs,and their stereoselectivity on many biological effects have been reported.Recent experiments have demonstrated that ginsenoside Rg3,which contains two neighbouring hydroxyl groups near and on the chiral centre C-20,can act as a natural ligand of PPAR?.An angiogenesis assay found that both Rg3 stereoisomers can induce angiogenesis effects via PPAR?,and the activity of 20(S)-Rg3 is stronger than that of 20(R)-Rg3.A fluorescence polarization and total internal reflection fluorescence(FP-TIRF)binding study also confirmed that only 20(S)-Rg3 can quantitatively bind to the PPAR?ligand-binding domain(PPAR?-LBD).As several reports have shown that synthetic PPAR? agonists,espeically rosiglitazone,may increase the risk of myocardial infraction and death from cardiovascular diseases,exploring a dietary supplement containing PPAR? agonist have great clinical significance.It is timely and of great interest to investigate the stereo-selective effects of Rg3 enantiomers on VSMCs proliferation and migration and atherosclerosis formation in diabetes.Objectives1.To verify whether PPAR-? is the downstream molecule of TFPI-2;2.To define the stereo-selective effects of Rg3 enantiomers on PPAR? activation;3.To explore the effects of differential PPAR? activation on AGEs-induced VSMCs proliferation and migration;4.To explore differential effects of Rg3 stereoisomers on atherosclerosis formation in diabetes,and the underlying mechanisms.Methods1.Cell culture and treatment(1)Human aortic smooth muscle cells(HASMCs,ATCC)were grown in Smooth Muscle Cell Medium containing 2%foetal bovine serum(FBS).HASMCs were infected with adenovirus vector mediated tissue factor pathway inhibitor-2 overexpression(OE-TFPI-2)and the negative control(OE-NC),mediun was changed overnight,then the cells were stimulated with AGEs(100?g/ml)for 48h.(2)Mouse vascular smooth muscle cells(MOVAS cells)and 293T cells were cultured in DMEM with 10%FBS.MOVAS cells were pretreated with 20(R/S)-Rg3(25?M,dissolved in 0.1%DMSO)in the presence or absence of GW9662(3?M)for 1h,and then stimulated with AGEs(100?g/ml)for 48h.An equal volume of DMSO was added to the controls.2.PPAR? reporter gene assayPPAR? reporter gene assay was performed in 293T cells transfected with reporter plasmid peroxisome proliferator-activated receptor response element(PPRE)X3-TK-luc,expression plasmids pSG5-PPAR? and pSG5-RXRa.After 6 hours of transfection,cells were washed once with Opti-MEM,supplemented by different concentrations of 20(S)-Rg3 or 20(R)-Rg3,further incubated with AGEs for 24 hours.Luciferase activity was measured using Luciferase Assay System with microplate reader.3.Cell proliferation assayProliferation was analysed by using the Cell Counting Kit-8 and Cell-LightTM EdU assay.4.Cell cycle analysisCell cycle analysis was performed by flow cytometry.5.Cell migration assayVSMC migration assays were performed in Transwell chambers.6.Gelatin zymographycell culture supernatants were electrophoresed in a 10%polyacrylamide gel containing 1 mg/ml gelatin.Lytic bands of gelatin digestion represented MMP2 and MMP9 activity.7.Animal modelEight-week-old male ApoE-/-mice were randomly divided into 6 groups:non-diabetic control group,DM(diabetic mellitus)+placebo group,DM+20(R)-Rg3 group,DM+ 20(R)-Rg3+GW9662 group,DM+20(S)-Rg3 group,and DM+20(S)-Rg3+GW9662 group.Type 1 diabetes mellitus was initiated by the administration of 5 daily intraperitoneal injections of 50 mg/kg streptozotocin(STZ);Mice with blood glucose levels of>300 mg/dL after 2 weeks were included in diabetic cohorts.Mice received a normal chow for the remaining 10 weeks.During the 6th-10th weeks,mice were given Rg3 at a dose of 10 mg/kg i.p.once two days,with oral gavage of GW9662 at 3 mg/kg per day.At the end,we monitored the blood glucose levels of the mice,then they were sacrificed.8.Biochemical measurementsSerum lipid profiles,including total cholesterol and triglyceride levels were measured by enzymatic assay with the use of an automatic biochemical analyser.9.Histology and immunohistochemistryEn face lesion staining with Oil-Red O was performed to assess overall burden and distribution of atherosclerosis.Cross sections of the aortic roots(predilection site of atherosclerosis).The sections were stained with hematoxylin and eosin(H&E).For immunofluorescence,the sections were labelled with ?-SMA and PCNA simultaneously.For immunohistochemistry,the sections were labelled with MMP2 and MMP9.10.Western blot analysisProteins were extracted from aorta of mice and treated cells,and the expressions of cyclinDl,cyclinE,PCNA MMP2,MMP9 and ?-actin were determined.11.Statistical analysisAll experiments were repeated at least three times,and data were presented as the mean ±S.E.M.Statistical analysis was carried out using ANOVA followed by Tukey's post hoc test(GraphPad Software,USA).P<0.05 was considered significant.Results1.PPAR? is the downstream molecule of TFPI-2TFPI-2 overexpression significantly up-regulated PPAR? expression,while TFPI-2 knockdown markedly suppressed PPAR? expression.Moreover,AGEs stimulation significantly down-regulated the expression levels of TFPI-2 and PPAR?,while TFPI-2 overexpression markedly reversed the decrease in PPAR? expression.2.Rg3 stereoisomers differentially activate PPAR?Rg3 treatment,especially 20(S)-Rg3,significantly increased luciferase activity in a dose-dependent manner(10-50 ?M).The PPAR? transcriptional activity as activated by 20(S)-Rg3 was more potent than 20(R)-Rg3.3.Effects of Rg3 stereoisomers on AGEs-induced VSMCs proliferationThe CCK-8 assay showed that Rg3 inhibited AGEs-induced proliferation of VSMCs in a concentration-dependent manner(10-50 ?M),and the effects of 20(S)-Rg3 was more potent than 20(R)-Rg3 at 25 ?M and 50 ?M.In Edu incorporation assay,20(R/S)-Rg3(25 ?M)both significantly reduced the stimulated DNA synthesis,and the effect of 20(S)-Rg3 was more potent,which could be abolished by PPAR? inhibition.4.Effects of Rg3 stereoisomers on AGEs-induced cell cycle progression in VSMCsIn AGEs-stimulated VSMCs,20(R/S)-Rg3 both significantly retarded cell cycle progression at G1 phase,and inhibited the increased expression of cyclinD1,cyclinE and PCNA.The effect of 20(S)-Rg3 was more potent than 20(R)-Rg3,both of which could be abolished by PPAR? inhibition.5.Effects of Rg3 stereoisomers on AGEs-induced VSMCs migrationIn AGEs-stimulated VSMCs,20(R/S)-Rg3 both significantly suppressed cell migration,and inhibited the increased expression and activity of MMP2 and MMP9.The effect of 20(S)-Rg3 was more potent than 20(R)-Rg3,both of which could be abolished by PPAR? inhibition.6.Biochemical parameters after Rg3 treatment in vivoTotal cholesterol and triglycerides levels did not differ among the diabetic ApoE-/-groups.However,there was a significant reduction in fasting glucose levels in the 20(S)-Rg3-treated group compared to the placebo-treated diabetic group.7.Effects of Rg3 stereoisomers on atherosclerotic plaque size and frequency of intraplaque VSMCsThe relative en face lesion area of the entire aorta and the cross-sectional plaque area of the aortic sinus were significantly reduced in the Rg3 treatment groups compared with the placebo-treated group and these effects were more significant in the 20(S)-Rg3 group than in the 20(R)-Rg3 group.8.Effects of Rg3 stereoisomers on the proliferation and migration of VSMCs within atherosclerotic plaquesCo-immunofluorescence staining indicated a marked reduction in the frequency of proliferating VSMCs(SMA+PCNA+)in the 20(S)-Rg3-treated group compared to that in the placebo-treated diabetic mice.Immunohistochemical analysis revealed that 20(S)-Rg3 markedly decreased the MMP2 and MMP9 intensity within atherosclerotic lesions,more potent than 20(R)-Rg3 treatment.In addition,Rg3 treatment,especially 20(S)-Rg3,significantly reduced the protein levels of cyclinD1,cyclinE,PCNA,MMP2 and MMP9 as compared with the placebo-treated group.Conclusion1.PPAR? is a downstream target of TFPI-2 in regulating VSMCs proliferation and migration.2.The effect of 20(S)-Rg3 on PPAR? activation was more potent than 20(R)-Rg3.3.Both 20(R/S)-Rg3 stereoisomers could inhibit AGEs-induced VSMCs proliferation and migration,but the effect of 20(S)-Rg3 was more potent due to stronger PPAR?activation,with a more remarkable G1 arrest and more significant downregulation of MMPs expression and activity.4.Rg3 could reduce the plaque size during de novo diabetic atherogenesis,secondary to reducing the proliferation and migration of VSMCs,and the effects of 20(S)-Rg3 was more potent due to stronger PPAR? activation.